The cellular redox homeostasis needs to be tightly regulated and controlled. This is achieved through a complex network of enzyme and antioxidant systems. Two of the major enzyme systems responsible for the maintenance of the cellular redox homeostasis are the thioredoxin system and the glutathione system. Glutathione (GSH) and glutathione reductase (GR) are collectively known as the glutathione enzyme system. GSH is the most abundant thiol-based antioxidant, found in millimolar concentrations within the cell—the oxidized form, GSSG, is reduced by the NADPH-dependent flavoenzyme GR. The classic mammalian thioredoxin system consists of two oxidoreductase proteins; thioredoxin (Trx) and thioredoxin reductase (TrxR) (Gromer, S., Urig, S., Becker, K., “The Thioredoxin System—From Science to Clinic”, Med. Res. Rev. (2004) 24, 40-89). There are three different thioredoxin isoenzymes encoded by separate genes; cytosolic thioredoxin (Trx1), thioredoxin located in the mitochondria (Trx2), and thioredoxin highly expressed in spermatozoa (SpTrx). The most studied thioredoxin is the classic Trx1, which is a ubiquitous 12 kDa cytosolic redox active protein.
All mammalian thioredoxins contain a conserved Cys-Gly-Pro-Cys-active site. The cysteine residues at positions 32 and 35 are key to the redox activity of Trx. Trx also contains 3 other cysteine residues at positions 62, 69 and 73. Although these cysteine residues do not form part of the active site, they are still essential for activity (Burke-Gaffney, A., Callister, M. E. J., Nakamura, H., “Thioredoxin: friend or foe in human disease?” Trends Pharm. Sci. (2005) 26, 398-404). It is thought that these three cysteine residues contribute to essential protein conformation, possibly through hydrogen bonding of the thiol group rather than through disulfide bond formation. Thioredoxin has many different physiological functions, but its main role appears to be maintaining cellular proteins in their dormant state (Holmgren, A., Arner, E. S. J. “Physiological functions of thioredoxin and thioredoxin reductase”, Eur. J. Biochem. (2000) 267, 6102-6109). For example, Trx inhibits the production of the transcription factor Nuclear factor κB (NFκB), by stabilizing NFκB, which is the inactive pre-cursor protein of NFκB.
Trx can act as a direct antioxidant, reducing hydrogen peroxide to water and oxygen. Peroxiredoxins also catalyse the reduction of hydrogen peroxide. Here, Trx reduces the oxidized peroxiredoxins and reactivates them. Trx also plays a part in regulating cellular apoptosis. This is achieved by Trx binding to apoptosis signaling kinase-1 (ASK-1) and maintaining it in its dormant state. Interestingly, this binding is lost when Trx is oxidized (Saitoh, M., Nishitoh, H., Fujii, M., Takeda, K., Tabiume, K., Sawada, Y., Kawabata, M., Miyazono, K., Ichijo, H, “Mammalian thioredoxin is a direct inhibitor of apoptosis signaling-regulating kinase (ASK) 1”, EMBO J. (1998) 17, 2596-2606). Extracellularly, Trx induces the chemotaxis of neutrophilic granulocytes, monocytes and T-cells.
Trx also has a role in DNA synthesis (Holmgren, A, “Thioredoxin and glutaredoxin systems”, J. Biol. Chem. (1989) 264, 13963-13966). Here, reduced Trx acts as a hydrogen donor for the enzyme ribonucleotide reductase, which is involved in the conversion of ribonucleoside 5′-diphosphates (NDP) to 2′deoxyribonucleoside 5′-diphosphates (dNDP), an essential step in DNA synthesis.
The other major component in the Trx system is thioredoxin reductase (TrxR), a NADPH-dependent enzyme that is responsible for reducing oxidized Trx via electron transfer through flavin adenine dinucleotide (FAD) as indicated in Reaction scheme 1. TrxR exists as a homodimer with one FAD and NADPH binding site per subunit.

The active site of TrxR contains a selenocysteine (SeCys) motif (Gladyshev, V. N., Jeang, K. T., Stadtman, T. C., “Selenocysteine, identified as the penultimate C-terminal residue in human T-Cell thioredoxin reductase, corresponding to TGA in the human placental gene”, Proc. Natl. Acad. Sci. USA (1996) 93, 6146-6151). In mammalian TrxR, the selenocysteine is located at the C-terminus, within the tetrapeptide Gly-Cys-SeCys-Gly-COOH. The selenocysteine residue is critical, as replacement of SeCys with cysteine (Cys) severely impairs TrxR activity (Zhong, L., Holmgren, A, “Essential role of selenium in the catalytic activities of mammalian thioredoxin reductase revealed by characterization of recombinant enzymes with selenocysteine mutations”, J. Biol. Chem. (2000) 275, 18121-18128). Another key residue involved in catalysis is the histidine residue located at position 472.
TrxR has a wide substrate specificity, most likely explained by the unique SeCys residue. Direct substrates for TrxR include dehydroascorbic acid, α-lipoic acid and hydrogen peroxide.
Thioredoxin primarily acts as an antioxidant in the cytoplasm when the cells redox homeostasis is intact, as previously discussed. When intracellular levels of reactive oxygen species (ROS) rise, Trx is translocated from the cytoplasm to the nucleus where it promotes the DNA binding of several transcription factors, including NFκB, AP-1 via Ref1, and p53 (Nordberg, J., Arner, E. S. J., “Reactive oxygen species, antioxidants, and the mammalian thioredoxin system”, Free Rad. Biol. Med. (2001) 31, 1287-1312).
Trx appears to play opposing roles in the regulation of NFκB, depending on the level of oxidative stress. At low levels of oxidative stress, Trx is predominantly found in the cytoplasm, where it blocks the degradation of the inactive IκB by interfering with the signals to IκB kinases. Under high levels of oxidative stress, Trx is predominantly found in the nucleus, where it enhances the ability of NFκB to bind to DNA. This is achieved by reduction of a critical cysteine residue identified as cysteine 62 of NFκB (Arrigo, A. P., “Gene expression and the thiol redox state”, Free Rad. Biol. Med. (1999) 27, 936-944). Once this residue is reduced, Trx is oxidized, which is then recycled by TrxR, to reduced Trx, allowing the process to reoccur (FIG. 1).
A number of organic compounds are known to inhibit the thioredoxin system. These include the compounds PX-12, AW464, Curcumin, Palmarumycin CP, and Pleurotin.

The majority of these compounds have been proposed as potential anticancer agents. For example, PX-12 has completed phase 1 clinical trials and entered phase 2 clinical trials for people suffering from advanced pancreatic cancer. Even though it was withdrawn early, due to lack of clinical efficacy linked to low Trx-1 levels in these tumours, its potential as an anti-cancer agent remains unchallenged (Ramanathan R K, Abbruzzese J, Dragovich T, Kirkpatrick L, Guilien J M, Baker A F, Pestano L A, Green S, Von Hoff D D, “A randomized phase II study of PX-12, an inhibitor of thioredoxin in patients with advanced cancer of the pancreas following progression after a gemcitabine-containing combination” Cancer Chemother Pharmacol. (2011) 67, 503-9). PX-12 has also been evaluated for the treatment of gastrointestinal cancers and, although this molecule was found to be unsuitable for intravenous infusion, the thioredoxin system remains a target for anticancer chemotherapy (Baker A. F., Adab K. N., Raghunand N., Chow H. H. Stratton S. P., Squire S. W., Boice M., Pestano L. A., Kirkpatrick D. L., Dragovich T., A phase IB trial of 24-hour intravenous PX-12, a thioredoxin-1 inhibitor, in patients with advanced gastrointestinal cancers, Invest. New Drugs, (2013) 31, 631-41.). Furthermore, it has been shown that AW464 can induce cellular apoptosis without increasing levels of oxygen free radicals (Stevens, M. F. G., Pallis, M., Bradshaw, T. D., Westwell, A. D., Grundy, M., Russell, N. “Induction of apoptosis without redox catastrophe by thioredoxin-inhibitory compounds”, Biochem. Pharmacol. (2003) 66, 1695-1705).
The known compounds are, however, subject to a number of limitations. For example, the metabolism of PX-12 after intravenous infusion leads to the production and exhalation of the noxious metabolite, butane thiol, and the known compounds appear to bind covalently to one or more enzymes of the thioredoxin system, which it is believed may cause increased toxicity. There is therefore a need to identify further inhibitors of the thioredoxin system.